Browsing by Author "Xiang, Jie"

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    Activation of Ftz-F1-Responsive Genes through Ftz/Ftz-F1 Dependent Enhancers
    (PLoS (Public Library of Science), 2016-10-10) Field, Amanda; Xiang, Jie; Anderson, W. Ray; Graham, Patricia; Pick, Leslie
    The orphan nuclear receptor Ftz-F1 is expressed in all somatic nuclei in Drosophila embryos, but mutations result in a pair-rule phenotype. This was explained by the interaction of Ftz-F1 with the homeodomain protein Ftz that is expressed in stripes in the primordia of segments missing in either ftz-f1 or ftz mutants. Ftz-F1 and Ftz were shown to physically interact and coordinately activate the expression of ftz itself and engrailed by synergistic binding to composite Ftz-F1/Ftz binding sites. However, attempts to identify additional target genes on the basis of Ftz-F1/ Ftz binding alone has met with only limited success. To discern rules for Ftz-F1 target site selection in vivo and to identify additional target genes, a microarray analysis was performed comparing wildtype and ftz-f1 mutant embryos. Ftz-F1-responsive genes most highly regulated included engrailed and nine additional genes expressed in patterns dependent on both ftz and ftz-f1. Candidate enhancers for these genes were identified by combining BDTNP Ftz ChIP-chip data with a computational search for Ftz-F1 binding sites. Of eight enhancer reporter genes tested in transgenic embryos, six generated expression patterns similar to the corresponding endogenous gene and expression was lost in ftz mutants. These studies identified a new set of Ftz-F1 targets, all of which are co-regulated by Ftz. Comparative analysis of enhancers containing Ftz/Ftz-F1 binding sites that were or were not bona fide targets in vivo suggested that GAF negatively regulates enhancers that contain Ftz/Ftz-F1 binding sites but are not actually utilized. These targets include other regulatory factors as well as genes involved directly in morphogenesis, providing insight into how pair-rule genes establish the body pattern.
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    Dermestes maculatus: an intermediate-germ beetle model system for evo-devo
    (Springer Nature, 2015-10-16) Xiang, Jie; Forrest, Iain S.; Pick, Leslie
    Understanding how genes change during evolution to direct the development of diverse body plans is a major goal of the evo-devo field. Achieving this will require the establishment of new model systems that represent key points in phylogeny. These new model systems must be amenable to laboratory culture, and molecular and functional approaches should be feasible. To date, studies of insects have been best represented by the model system Drosophila melanogaster. Given the enormous diversity represented by insect taxa, comparative studies within this clade will provide a wealth of information about the evolutionary potential and trajectories of alternative developmental strategies. Here we established the beetle Dermestes maculatus, a member of the speciose clade Coleoptera, as a new insect model system. We have maintained a continuously breeding culture in the lab and documented Dermestes maculatus embryogenesis using nuclear and phalloidin staining. Anterior segments are specified during the blastoderm stage before gastrulation, and posterior segments are added sequentially during germ band elongation. We isolated and studied the expression and function of the pair-rule segmentation gene paired in Dermestes maculatus. In this species, paired is expressed in stripes during both blastoderm and germ band stages: four primary stripes arise prior to gastrulation, confirming an intermediate-germ mode of development for this species. As in other insects, these primary stripes then split into secondary stripes. To study gene function, we established both embryonic and parental RNAi. Knockdown of Dmac-paired with either method resulted in pair-rule-like segmentation defects, including loss of Engrailed expression in alternate stripes. These studies establish basic approaches necessary to use Dermestes maculatus as a model system. Methods are now available for use of this intermediate-germ insect for future studies of the evolution of regulatory networks controlling insect segmentation, as well as of other processes in development and homeostasis. Consistent with the role of paired in long-germ Drosophila and shorter-germ Tribolium, paired functions as a pair-rule segmentation gene in Dermestes maculatus. Thus, paired retains pair-rule function in insects with different modes of segment addition.
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    INVESTIGATING PAIR-RULE GENE ORTHOLOGS IN AN INTERMEDIATE GERM BEETLE, DERMESTES MACULATUS
    (2017) Xiang, Jie; Pick, Leslie; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Insects share a body plan based on repeating segments. Segmentation has been well characterized in Drosophila melanogaster, in which segments are established by a genetic hierarchy including gap, pair-rule and segment polarity genes. Pair-rule genes (PRGs) are a key class of segmentation genes as they are the first cohort of genes expressed in a periodic pattern. Segments are established simultaneously in Drosophila in early embryos, while most other insects add segments sequentially as the embryo elongates. Our goal is to understand molecular mechanisms controlling segment formation and to determine the extent of their conservation during evolution. Here, we established the hide beetle Dermestes maculatus, an intermediate germ developer, as a new model system for studying segmentation patterning. We first established a lab colony and studied early embryogenesis in Dermestes. All nine PRG orthologs were isolated using degenerate PCR and RACE, and their expression patterns were examined with in situ hybridization. Except for opa, all Dermestes PRG orthologs are expressed in PR-like striped patterns. Gene functions were tested using RNA interference (RNAi). We examined both hatched and unhatched larvae to uncover defects with different severities. Both Dmac-prd and -slp knockdown resulted in typical PR defects, suggesting that they are “core” PR genes. Dmac-eve, -run and -odd have dual roles in germ band elongation and in PR segmentation, as severe knockdown caused anterior-only, asegmental embryos while moderate knockdown resulted in PR-like defects. Elongated but asegmental germ bands resulted from Dmac-prd and -slp double knockdown, suggesting decoupling of germ band elongation and PR segmentation. Extensive cell death prefigured the cuticle patterns after knockdowns, seen long ago for Drosophila PR phenotypes, although disrupted cell mitosis was also observed after Dmac-eve knockdown. We propose that PRGs have retained basic roles in PR segmentation during the transition from short-to-long germ development and share evolutionary conserved functions in promoting cell viability. Finally, I also present detailed protocols on Dermestes lab rearing, embryo collection and fixation, in situ hybridization and RNAi. The technical information described here will provide useful information for other genetic studies in this new model system.